Recent studies have indicated the presence of new NADPH oxidase (Nox) homologs within the airways, with homology to phagocytic gp91phox (Nox2), that are responsible for apical epithelial production of H2O2 in response to various inflammatory or environmental stimuli. These Nox homologs, termed Dual Oxidases (Duox), exist as two isoforms, of which Duox1 is primarily expressed in the tracheobronchial epithelium, whereas Duox2 has been detected in salivary or submucosal glands. In addition to postulated roles in airway host defense, recent studies have suggested alternative functions of airway epithelial Duox1, including regulation of epithelial H+ transport and involvement in epithelial responses to injury by promoting production of growth factors, metalloproteinases, and cytokines, and stimulating epithelial cell migration and repair. Our recent studies have indicated Duox-mediated H2O2 production in tracheobronchial epithelial cells in response to mechanical injury, which is mediated by cellular release of ATP and stimulation of purinergic P2 receptors at the epithelial surface. Moreover, epithelial cell migration and wound repair were found to be mediated by ATP- mediated activation of mitogen-activated protein kinase (MAPK) pathways and activation of metalloproteinases including matrix metalloproteinase (MMP)-9, by mechanisms involving Duox1. Therefore, we hypothesize that Duox1 contributes to maintenance of airway epithelial barrier integrity, by stimulating epithelial repair processes in response to injury, by localized oxidative events at the epithelial surface and/or by activation of redox-dependent cellular signaling pathways. In addition, based on recent observations that Th2 cytokines (IL-13, IL-4) can induce epithelial Duox1 expression, and that lung Duox1 expression is markedly increased in a mouse model of allergic airway inflammation, we propose that exaggerated or persistent Duox1 activation may contribute to a chronic wound response and airway remodeling, as is observed in chronic asthma. The main objectives of this proposal are to identify the mechanisms by which Duox1 activation mediates epithelial cell migration and repair in vitro, and to establish the contribution of Duox1 to airway epithelial repair and remodeling in vivo. We will determine the contribution of extracellular or cellular H2O2 production and/or localized pH changes and H+ transport to Duox1-mediated epithelial cell migration (Aim 1), identify mechanisms involved in Duox1-dependent MAPK activation and growth factor/MMP activation (Aim 2), and characterize extracellular and cellular redox-sensitive targets of Duox1-derived H2O2 by redox proteomics approaches (Aim 3). Finally, we will investigate the contribution of Duox1 to epithelial repair in a model of epithelial injury using naphthalene, and in a mouse model of allergic airway inflammation (Aim 4). Collectively, these studies will provide important new insights into the epithelial biology of Duox1, and will establish the potential contribution of Duox in airway remodeling during chronic airway diseases such as asthma. PUBLIC HEALTH RELEVANCE. The airway epithelium is in continuous contact with the environment and is critical in lung defense against inhaled toxins and microbial organisms. In chronic airway diseases such as asthma, the airway epithelium is injured and mechanisms that stimulate epithelial cell growth and repair are activated. Recent studies have identified the presence of an oxidant-producing enzyme, Duox1, within the airway epithelium, which may contribute to airway host defense, similar to recent enzymes in phagocytes. However, our recent studies have also demonstrated that Duox contributes to epithelial repair processes after injury. Thus, although oxidative stress is believed to contribute to tissue damage during disease, low levels of oxidant production by epithelial Duox1 may be beneficial in maintaining airway epithelial barrier integrity. The goal of this project is to investigate the molecular mechanisms by which Duox1 activation mediates epithelial repair processes, and how cellular oxidants produced by Duox1 contribute to this. Secondly, we will investigate the importance of Duox1 in epithelial repair in in vivo models of epithelial injury and allergic airway disease such as asthma. Collectively, these studies will further our understanding of the biological roles of airway Duox1, and its potential importance in chronic airway diseases such as asthma. [unreadable] [unreadable] [unreadable]